Oil Palm Bulletin 53 (November 2006) p. 36 - 48 Biomarkers...

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* Malaysian Palm Oil Board, P. O. Box 10620, 50720 Kuala Lumpur, Malaysia.

Oil Palm Bulletin 53 (November 2006) p. 36 - 48

ABSTRACT

The poor embryogenesis rate in oil palm tissue culture has ignited interest in the study of somatic embryogenesis. There is a need to understand the mechanisms involved in determining the embryogenic potential of the material in culture. Research was tailored to initially elucidate the differences between embryogenic calli (EC) and non-embryogenic calli (NEC). As both EC and NEC were found to be distinctly different, this implies that their gene expressions are differentially regulated as well. Studies in other areas have successfully used gene expression patterns to classify and predict outcomes of specific conditions. This then led to a concerted effort to isolate and characterize the genes related to somatic embryogenesis. Part 1 of this paper basically concentrates on detailing some of the differences found between embryogenic cultures and their non-embryogenic counterpart.

ABSTRAK

Kadar embriogenik yang rendah dalam kultur tisu sawit telah membangkitkan minat untuk mengkaji embriogenesis somatik. Pemahaman tentang mekanisme yang terlibat dalam mengenal pasti potensi embriogenik bahan dalam kultur amat diperlukan. Penyelidikan pada peringkat awal adalah untuk menjelaskan perbezaan yang terdapat antara kalus embriogenik (EC) dan kalus tak embriogenik (NEC). Kedua-dua EC dan NEC didapati mempunyai ciri-ciri yang berbeza. Ini mencadangkan agar pengekspresan gen EC dan NEC juga dikawalatur secara berbeza. Kajian dalam bidang penyelidikan lain telah berjaya menggunakan pola pengekspresan gen untuk mengklasifikasi dan meramal hasil sesuatu keadaan yang spesifik. Oleh itu, usaha untuk memencil dan mencirikan gen yang terlibat dalam proses embriogenesis somatik telah ditingkatkan. Bahagian 1 artikel ini memberi tumpuan pada perbezaan penghuraian yang terdapat pada kultur embriogenik dan tak embriogenik.

Biomarkers:FindingaNicheinOilPalmTissueCulture.Part1–LayingtheFoundationMeilinaOng-Abdullah*andOoiSiewEng*

Keywords: o�l palm, embryogen�c call� (EC), somat�c embryogenes�s, gene express�on, b�omarkers.

INTRODUCTION

Much of the 1990s were spent understand�ng the floral abnormality phenomenon, better known as mantl�ng. Efforts were channelled �nternally as well as externally to d�ssect the problem and study �t from d�fferent angles to unravel the causes of th�s cond�t�on. However, there was yet another setback �n an otherw�se very v�able propagat�on system for the o�l palm. W�th research on abnormal�ty tak�ng centre stage, th�s other problem lurk�ng �n the t�ssue culture laborator�es was not g�ven equal emphas�s. As t�ssue culture laborator�es gathered more data, �t was revealed that the overall average rate of embryogenes�s of cultures per se ar�s�ng from the convent�onal sol�d culture system was only approx�mately 6% (Woo�, 1995)! Desp�te �ts obv�ous �mportance, l�ttle �s known about the b�ology of somat�c embryogenes�s of the o�l palm.

Most of the earl�er stud�es done on somat�c embryogenes�s of the o�l palm concentrated on the development of methodolog�es for the �n�t�at�on and product�on of somat�c embryos (Jones, 1974; Ahee et al., 1981; Pannet�er et al., 1981). These groups ma�nly man�pulated phytohormones �n the med�a compos�t�on to determ�ne the t�ssues w�th better clonab�l�ty to �mprove the process. In 1988, Schwend�man et al. carr�ed out a h�stolog�cal analys�s of somat�c embryogenes�s from leaf-der�ved callus deta�l�ng the emergence of callus and the subsequent format�on of somat�c embryos, w�th shoot and root ap�ces. Not long before that, Turnham and Northcote (1982) had �nvest�gated the occurrence of b�ochem�cal �nd�cators that are useful �n the pred�ct�on of embryogen�c potent�al. They showed �t was poss�ble to correlate the act�v�ty of acetyl-CoA carboxylase w�th the degree of embryogenes�s �n o�l palm cultures.

W�th the advent of molecular b�ology, the understand�ng of molecular sw�tches that occur �n somat�c cells to �nduce them to become embryogen�c has ga�ned �mportance (Dud�ts et al., 1995). It �s believed that signaling systems via the influences of var�ous stresses appl�ed preferent�ally externally by hormones play an essent�al role �n the �nduct�on of

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cell d�v�s�on dur�ng generat�on of the embryogen�c stage.

It �s now common knowledge that poly embryo�d (PE) cultures, ma�nta�ned for prolonged per�ods v�a subcultur�ng, show a h�gher frequency of abnormal�ty (Corley et al., 1986). As the frequency and sever�ty of abnormal�t�es �ncrease w�th culture t�me, by l�m�t�ng or reduc�ng the number of culture cycles the occurrences can be m�n�m�zed. However, th�s w�ll also reduce the number of shoots produced. Thus, it is vital to increase the efficiency of callusing, embryogenes�s, germ�nat�on and prol�ferat�on of embryo�ds so that the number of subcultures can be reduced w�th less loss �n shoot generat�on and, consequently, plantlet product�on. It can be argued that w�th the advent and rap�d �mprovement of the l�qu�d culture system (Wong et al., 1999; Tarm�z� et al., 2003; 2005) as a far more efficient micropropagation system, coupled w�th good select�on and laboratory pract�ces, the problems of poor embryogenes�s as well as abnormal�ty can be cons�dered problems of the past. However, th�s does not d�sm�ss the fact that t�ssue culture �s often more art than sc�ence. It �s our �ntent�on to �nfuse the sc�ence w�th some art and so scientifically explain, at least partially, what happens to the plant mater�al as �t goes through the stages �n the o�l palm t�ssue culture process.

Th�s paper h�ghl�ghts the work done thus far �n our laboratory �n embryogenes�s. Research �s geared towards understand�ng and �dent�fy�ng the underly�ng factors �nvolved �n the �nduct�on of embryogenes�s. In the early work, attempts were made to ascerta�n the d�fferences between embryogen�c and non-embryogen�c cultures. Th�s led to a study of the embryogenes�s process at the molecular level wh�ch la�d the groundwork for the subsequent �solat�on of potent�al markers for embryogenes�s.

EMBRYOGENESIS

Embryogenes�s �n ang�osperms w�th Arabidopsis as a model d�cot system, beg�ns w�th a double fert�l�zat�on event �n wh�ch two sperm nucle� fuse w�th the egg cell and central cell nucle� to �n�t�ate embryo and endosperm development, respect�vely. The zygote then undergoes a ser�es of cell d�v�s�ons and d�fferent�at�on events to produce the mature embryo (West and Harada, 1993; Laux and Jurgens, 1997).

The first zygotic division is asymmetric, giving r�se to a small ap�cal cell and a large basal cell. These cells have d�fferent fates, w�th the ap�cal cell go�ng on to produce the embryo proper and the basal cell generat�ng the hypophys�s and the suspensor, a trans�ent organ that plays both structural and

phys�olog�cal roles dur�ng embryo development. As the ap�cal cell d�v�des, a structure w�th e�ght �sod�ametr�c cells �s formed, known as the octant stage. At th�s stage, the cells of the embryo proper are organ�zed �n two t�ers - the upper t�er dest�ned to form the cotyledons and shoot apex, and the lower w�ll develop the hypocotyl. An `O’ boundary separates the two t�ers. Per�cl�nal cell d�v�s�ons within the octant stage embryos generate the first embryon�c t�ssue, the protoderm, wh�ch �s the precursor of the ep�derm�s. The del�neat�on of the protoderm establ�shes the globular stage embryo. The embryo �ncreases �n s�ze and cell number by ant�cl�nal cell d�v�s�ons of the protoderm followed by long�tud�nal and transverse d�v�s�ons of the �nter�or cells. These later d�v�s�ons w�th�n the globular embryo produce elongated cells �n the centre of the embryo that defines the procambium and ground mer�stem. Also, at th�s stage, the uppermost cells of the suspensor undergo d�v�s�on to generate the hypophys�s that g�ves r�se to part of the qu�escent centre and the �n�t�als of the central root cap. Dur�ng trans�t�on from the globular stage to the heart stage of embryogenes�s, local�zed cell d�v�s�ons generate the cotyledons and, by the torpedo stage, both the shoot and root ap�cal mer�stems are v�s�ble as organ�zed structure. These processes are dep�cted �n Figure 1.

A mature dicot embryo consists of at least five major organs/structures along �ts ap�cal-basal ax�s: shoot ap�cal mer�stem, cotyledons, hypocotyl, root and root ap�cal mer�stem. The rad�al ax�s of the embryo cons�sts of three pr�mary t�ssue systems: outer protoderm, m�ddle ground mer�stem and �nner procamb�um (vascular t�ssue).

The whole process descr�bed above makes up the first phase of embryogenesis in higher plants. Accord�ng to Me�nke (1991), cell d�v�s�on and morphogenes�s character�ze the early stages of the plant embryo development. In the phase follow�ng th�s, the embryo goes through maturat�on, character�zed by the accumulat�on of storage reserves. F�nally, the embryo prepares for des�ccat�on, becomes des�ccated and enters a per�od of developmental arrest (West and Harada, 1993).

In compar�son to the�r d�cot counterpart, monocots have str�k�ngly d�fferent mature embryos because of hav�ng only one cotyledon. However, the development of the embryo up to the octant stage �n both d�cots and monocots are almost �dent�cal (Raghavan and Sharma, 1995). In monocots, the globular stage then develops �nto a trans�t�on-state embryo (heart shape embryos are not observed) wh�ch conta�ns an �ntegral suspensor. A scutellum �s formed laterally at th�s trans�t�on state embryo, and shoot and root pr�mord�a develop at both ends

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of the embryo ax�s. The scutellum subsequently develops �nto the s�ngle cotyledon (Toonen and de Vr�es, 1996).

ZYGOTICEMBRYOGENESISvERSUSSOMATICEMBRYOGENESIS

Fert�l�zat�on events and subsequent embryo development normally occur deep w�th�n the maternal t�ssues. Furthermore, the early embryo �s m�nute and �s surrounded by both endosperm and maternal cells. Th�s phys�cal �naccess�b�l�ty greatly hampers analys�s of the early developmental events dur�ng embryogenes�s. As a consequence, very l�ttle �s known about the genes �nvolved �n early embryogenes�s and the�r regulat�on �n h�gher plants. However, �ntens�ve efforts to genet�cally �dent�fy the genes requ�red �n early embryogenes�s through mutant stud�es �n model systems such as Arabidopsis and ma�ze (West and Harada, 1993) have prov�ded �ns�ghts �nto the molecular mechan�sms

of embryogenes�s. These analyses would be greatly enhanced w�th the ava�lab�l�ty of an appropr�ate in vitro model that �s not l�m�ted �n t�ssue quant�ty and access�b�l�ty, the �deal one of wh�ch �s the somat�c embryo system (Z�mmerman, 1993).

The obv�ous d�fference between zygot�c and somat�c embryogenes�s �s that the latter ut�l�zes the plants un�que capab�l�ty to produce morpholog�cally and developmentally normal embryos that would progress �nto whole plants, from und�fferent�ated somat�c cells �n culture. In short, somat�c embryogenes�s �s the format�on of embryos from cells other than gamet�c cells or through gamet�c fus�on (Steward, 1958). Although the phys�cal and chem�cal env�ronment surround�ng both the somat�c and zygot�c embryos are d�fferent, fasc�nat�ngly, they share s�m�lar developmental patterns.

Both zygot�c and somat�c embryogenes�s are generally known to be complex processes and they

Figure 1. An overview of plant embryogenesis. T, terminal cell; B, basal cell; EP, embryo proper; S, suspensor; Bc, suspensor basal cell; Pd, protoderm; u, upper tier; l, lower tier; Hs, hypophysis; Pc, procambium;

Gm, ground meristem; C, cotyledon; A, axis; MPE, micropylar end; CE, chalazal end; SC, seed coat; En, endosperm; SM, shoot meristem; RM, root meristem.

Biomarkers: Finding a Niche in Oil Palm Tissue CultureOil Palm Bulletin 53

Zygote 2-cell2/4-cell

EP 8-cellEP 16-cell

EP Globularembryo

Trans�t�onembryo

Heartembryo

C

A

Organ expans�on and maturat�on

CESC

En

SM

RM

MPETorpedo embryo Walk�ng-st�ck embryo Mature embryo

SM

SC

Pd

GmPc

En

A

RM

S

Source: Goldberg et al. (1994).

Post-fert�l�zat�on Global heart trans�t�on

Pro-embryo

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have been w�dely descr�bed �n the l�terature (Me�nke, 1995). In contrast to the zygote, wh�ch �s �ntr�ns�cally embryogen�c, somat�c embryogenes�s requ�res �nduct�on of embryogen�c competence �n cells wh�ch are not naturally embryogen�c. Furthermore, the acqu�s�t�on of embryogen�c competence �nvolves an �nduct�on phase for wh�ch there �s no d�rect counterpart �n zygot�c embryogenes�s (Dodeman et al., 1997).

Here are some of the compar�sons frequently made between zygot�c and somat�c embryogenes�s:

Asymmetric Cell Division and Polarity

Hyman and Stearns (1992) establ�shed that the prerequ�s�te for �nduct�on of cell d�fferent�at�on �s cell polar�ty followed by asymmetr�c cell d�v�s�on. In ang�osperms, polar�ty �n both the female gamete and the zygote seems const�tut�ve, �nd�cat�ng pre-determination of the first division phase. With identification and isolation of the genes controlling the format�on of zygot�c embryos �n d�fferent systems, more l�ght has been shed on th�s process (Nagato et al., 1989; Jurgens et al., 1991; Me�nke, 1991, Clark and Sher�dan, 1991; Mayer et al., 1993; We�gel, 1993; K�tano et al., 1993). However, Me�nke (1995) found it difficult to distinguish between housekeep�ng and regulatory funct�ons �n plant embryogenes�s. Th�s �s because �mportant genes found �n embryogenes�s are often also expressed �n vegetat�ve t�ssues, an example of th�s be�ng the gnom mutant. The GNOM gene not only affects early events �n plant morphogenes�s (Lloyd, 1991) but has also been found to be �nvolved �n a secretory pathway (Shevell et al., 1994). Thus, the GNOM gene may affect the synthes�s and secret�on of components, such as glycoprote�ns, requ�red for proper cell d�v�s�on, cell elongat�on and cell-to-cell contact.

Cell polar�ty and asymmetr�c cell d�v�s�on are also �nvolved �n the �n�t�at�on of somat�c embryogenes�s. It �s known that plant cells respond to a var�ety of env�ronmental and cellular cues. One of the more popularly used st�mulat�on are the growth regulators. Aux�ns have been found to promote asymmetr�c d�v�s�on and to be the med�ator �n the trans�t�on from somat�c to embryogen�c cells (Bogre et al., 1990; Komam�ne et al., 1990; Dud�ts et al., 1991). Bes�des aux�ns, cytok�n�ns can also �nduce changes to d�v�s�onal planes, result�ng �n the format�on of embryogen�c cells (Maheswaran and W�ll�ams, 1985). The presence of exogenous growth regulators probably could have interfered with pH gradients or electrical fields around the cells, hence causing modifications to cell polar�ty (D�jak et al., 1986; Sm�th and Kr�kor�an, 1990).

To summar�ze, �n both zygot�c and somat�c embryogenes�s, controlled cell expans�on and asymmetr�c d�v�s�ons are �mportant mechan�sms �n the format�on of embryogen�c cells. They are also l�nked to the heterogenous part�t�on�ng of cytoplasm�c determ�nants subsequent to the format�on of cell polar�ty, wh�ch seems to be a prerequ�s�te for the �n�t�at�on of embryogenes�s.

Pattern Formation

The pattern format�on �n the zygote embryo is very well conserved with the first asymmetric d�v�s�on followed by the octant stage, then format�on of the protoderm and �n�t�at�on of the pr�mord�a. There are apparent �nd�cators show�ng the �n�t�al patterns of somat�c and zygot�c embryos to be d�fferent. For example, somat�c embryos of tobacco, and even o�l palm, are much larger than the�r zygot�c counterparts (Stolarz et al., 1991). However, g�ven the prec�s�on of the zygot�c embryogenes�s programme, �t would be expected that somat�c embryogenes�s w�ll follow the same pattern. Th�s was supported by van Engelen and de Vr�es (1992) who were able to show that the first stages of both zygot�c and somat�c embryos can be �dent�cal �f not at least very s�m�lar to each other.

However, there are some d�fferences between them. Somat�c embryos do not have an endosperm and suspensor, wh�ch are �mportant elements �n zygot�c embryos (Dodeman et al., 1997).

Meristem Formation

The format�on of the mer�stem �n zygot�c embryos has been stud�ed �n much deta�l through funct�onal mutants. In somat�c embryogenes�s, most of the abnormal�t�es d�splayed by the embryos are also found �n zygot�c embryos, for example, aberrant embryos of the grapev�ne cult�var 41B w�thout a funct�onal mer�stem show s�m�lar h�stolog�cal patterns as the SHOOT MERISTEMLESS mutant (Goebel-Tourand et al., 1993). Accord�ng to Goebel-Tourand et al., �n controll�ng embryo development, hormonal balance �s of the utmost �mportance.

Maturation and Germination

Dur�ng rap�d d�fferent�at�on and organogenes�s, no storage prote�ns are accumulated. However, once these stages are completed, an �ncreased rate of synthes�s and depos�t�on of storage prote�ns, l�p�ds and starch occur, result�ng �n cell expans�on.

In zygot�c embryos, the accumulat�on of storage prote�ns (Galau et al., 1991) �s followed by accumulat�on of late abundant (LEA) prote�ns, some of wh�ch have been shown to be ABA-�nduced


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and may part�c�pate �n des�ccat�on tolerance (Dure et al., 1989). Stud�es on storage prote�n synthes�s �n somat�c embryos of cotton at the globular stage showed that the accumulat�on patterns observed �n somat�c embryos are s�m�lar to those reported �n zygot�c embryos except that they appear at an earl�er stage and the prote�ns at much lower concentrat�on (Shoemaker et al., 1987). In another �nstance, storage prote�ns, 7S, 11S and 2S, �n somat�c embryos of alfalfa also accumulated �n the�r zygot�c embryos, although the�r k�net�cs of synthes�s are d�fferent (Krochko et al., 1992).

In general, somat�c embryos develop through stages s�m�lar to those of zygot�c embryos except that they do not become dormant. In add�t�on to th�s, the �ntegument and endosperm, wh�ch are requ�red for conservat�on and germ�nat�on, respect�vely, are absent �n somat�c embryos (Dodeman et al., 1997).

EMBRYOGENICCALLI(EC)vERSUSNON-EMBRYOGENICCALLI(NEC)

In response to hormonal treatments (�n general aux�ns), explant cells w�ll usually ded�fferent�ate �nto callus t�ssue, wh�ch then undergo e�ther organogenes�s or embryogenes�s. Callus t�ssues that have acqu�red an organogenet�c capac�ty are able to regenerate organs, usually shoots that, upon transfer to a d�fferent hormonal cond�t�on, would complete the format�on of the rad�cal system (Lo Sch�avo et al., 1989). However, �n the case of embryogenes�s, these cells are comm�tted to an embryogen�c fate. Th�s embryogen�c state, also known as the ground state (State 0) of plant development (Figure 2), �n the presence of aux�n, can generate pro-embryogen�c masses (PEM) and then embryos with high efficiency (Sung et al., 1984; Nomura and Komam�ne, 1985).

S�m�lar to r�ce, ma�ze and herbaceous monocots (Chen and Luthe, 1987; Rao et al., 1990), o�l palm ECs are heterogeneous �n nature and conta�n some non-embryogen�c cells. NECs are thought to ar�se e�ther from the d�v�s�on of non-�nduced cells present �n the or�g�nal explant or from the d�fferent�at�on of embryogen�c cells, caused by decl�n�ng levels of exogenous plant growth regulators �n the med�um through the�r rap�d metabol�sm and/or degradat�on. The rap�d removal of non-embryogen�c cells or sectors through subculture �s useful for the ma�ntenance of embryogen�c cultures (A Ruslan, pers. comm.).

The morpholog�cal character�st�cs of the o�l palm ECs seem to conform to the group of Type II call� (Green et al., 1983), �n contrast to NECs wh�ch are Type I. PEs are d�st�nctly found only �n ECs and the�r presence may be an �nd�cator for embryogen�c potent�al. It �s not surpr�s�ng that NECs, devo�d of

PEs, lack th�s potent�al. It was observed that one to several layers of large and h�ghly vacuolated cells usually surround these PE structures. St�ll on the same subject, aux�ns have been �mpl�cated �n the cause of callus fr�ab�l�ty due to aux�n-�nduced cell separat�on that eventually leads to �solat�on of cells and groups of cells (Evans et al., 1981). Th�s can be observed �n Figure 3 w�th the format�on of proembryos, wh�ch usually have a th�ckened cell wall (arrow).

As seen dur�ng the format�on of reproduct�ve structures, such as the embryo sac and zygote, plasmodesmatal connect�ons are usually absent w�th no d�rect symplast�c connect�on between the zygote and �ts maternal t�ssues (Yeung, 1995). Based on th�s, phys�olog�cal �solat�on has been suggested, that �s, absence of plasmodesmata and, hence, symplast�c transport �n somat�c embryogenes�s. However, whether th�s isolation �s a prerequ�s�te for embryogen�c cell �nduct�on �s moot. At least �n the o�l palm, the format�on of PEs surrounded by several layers of h�ghly vacuolated cells w�th shrunken cytoplasm seem to support the necess�ty of the �solat�on for embryogenes�s.

Plasmodesmatal connect�ons have been d�st�nctly observed �n cells w�th�n the pro-embryo masses (Figure 3b). Accord�ng to Jas�k et al. (1995), these connect�ons are only present �n organ�zed cells. The layers of cells w�th severed plasmodesmata that separate the pro-embryos from other ne�ghbour�ng cells act as a barr�er to create the cond�t�on of isolated groups of cells. Th�s allows the �solated cells to d�fferent�ate and develop, free of any influence from their surrounding cells, into organ�zed structures. Th�s emphas�zes the fact that the presence of these layers of h�ghly vacuolated cells �s just as �mportant as the format�on of pro-embryos dur�ng embryogenes�s. In fact, the former has to occur before any pro-embryo masses can be detected �n embryogen�c cultures (S H Mantell, pers. comm.). In add�t�on to th�s, depos�t�on of callose, a β 1-3-l�nked glucan, has also been suggested to be a prerequ�s�te for a cell to undergo a new metabol�c programme because each t�me callose depos�t�on around ord�nary cells �s st�mulated, �t �s usually followed by enhanced somat�c embryogenes�s (Dubo�s et al., 1990). Callose depos�ts can seal off the plasmodesmata and block the connect�ng strands between s�eve elements, thus �solat�ng groups of cells or reg�ons of t�ssues from the greater symplasm (Romberger and Hejnow�cz, 1993).

At the ultrastructural level, m�tochondr�a are often located near organelles that consume energy, w�th the�r number determ�ned by the level of energy needed by the cell (Warren and W�ckner, 1996). Th�s observat�on �s �n l�ne w�th phys�olog�cal results that

Biomakers: Finding a Niche in Oil Palm Tissue CultureOil Palm Bulletin 53

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Figure 3. (a) Histological section of EC showing formation of pro-embryos (pe) which is usually accompanied by thickening of surrounding wall (arrowhead). Magnification: 100x. (b) Transmission electron micrograph (TEM) showing the presence of plasmodesmatal connections (pd) in EC. Magnification: 10K x ZOOM, 75kV. (c) and (d) An ultrastructural comparison between EC and NEC. Magnification: 5K x ZOOM, 75kV. Pro-embryo, pe;

plasmodesmata, pd; cell wall, cw; mitochondria, mt; endoplasmic reticulum, er; vacuole, va; nucleus, nu.

Figure 2. Developmental phases in somatic embryogenesis of carrot suspension cultures.


Source: Koman�ne et al. (1992).

pe

pe

pe

pd

cwmt

mt

nu

er pd va

mtnu

42

ECs exh�b�t h�gher resp�rat�on rate than the�r non-embryogen�c counterpart (data not shown). Th�s �s reflected by the fact that changes in the composition or abundance of organelles �n a t�ssue �s governed by the�r cell type and funct�on (Nunnar� and Walter, 1996). In order for the cells to respond to cellular and organ�smal needs, there must be an �nvolvement of �ntracellular commun�cat�on pathways controll�ng gene express�on programmes �n the t�ssues.

Resp�rat�on �s cons�dered to be of very large flux, and to be tightly connected to many processes �n the plant system, such as growth, allocat�on (espec�ally for ma�ntenance), n�trogen uptake, etc. It �s also very �mportant for the determ�nat�on of net pr�mary product�on and plant death (Cannell and Thornley, 2000). In plants, resp�rat�on �s usually t�ed up w�th photosynthes�s; however �n in vitro cultures photosynthes�s can be d�sregarded as a carbon (C) source �s �ncorporated �n the culture med�um.

Accord�ng to Warren and Fowler (1978), the doubl�ng t�me of a cell cycle of cultured carrot cells decreases when the cells are �nduced to undergo embryogenesis. This signifies that the cells have a rap�d metabol�c rate as energy �s needed for cell d�v�s�on and d�fferent�at�on, wh�ch eventually leads to the development of the culture as a whole. Where there �s rap�d metabol�sm, resp�rat�on would be expected to �ncrease proport�onately. Th�s �s because resp�rat�on �s controlled by the energy demands of the cells as well as the supply of C substrates (Farrer, 1985; Amthor, 1994). If the C supply �s not l�m�t�ng, then resp�rat�on would be pos�t�vely correlated w�th the overall demand for ATP and NAD(P)H. Although th�s �s the case for resp�rat�on �n photosynthet�c plant, �t seems log�cal that �t also holds true for the in vitro system. M�tochondr�al resp�rat�on consumes C subtrates (mostly glucose) to prov�de energy as ATP and reduc�ng power [NAD(P)H] for all the energy requ�r�ng processes �n plants (Cannell and Thornley, 2000). Th�s br�ngs to m�nd the TEM observat�on of EC earl�er �n th�s sect�on. The m�tochondr�a were more and denser �n the EC than NEC (Figures 3c and d). And as ment�oned by Nunnar� and Walter (1996), cells have d�fferent compos�t�ons or abundance of organelles because of the�r d�fferent cell types and/or funct�ons. Th�s supports the observat�on that EC and SC go through more rap�d resp�rat�on (based on the product�on of CO2) than NEC.

HORMONALREGULATIONDURINGEMBRYOGENESIS

It �s now known that EC and NEC are character�st�cally very d�fferent; however, the most intriguing question is, why are only specific tissues w�th�n the or�g�nal explant capable of form�ng

embryogen�c callus? Rajasekaran et al. (1987), from the�r work on Pennisetum purpureum, reported that d�fferent levels of endogenous growth substances �n d�fferent reg�ons of the leaves correlated w�th the�r regenerat�ve potent�al when cultured. Th�s �nd�cates that �n acqu�r�ng competence to respond to an �nduct�ve s�gnal, the d�fferent�ated state of the explant �s �mportant. Th�s �s regarded as the State 0 cells (Figure 2) whereby they are able to respond to external cues (e.g. aux�n) wh�ch are necessary for cell competency �n order to become tot�potent (Komam�ne et al., 1992). And once th�s �s ach�eved, st�ll �n the presence of aux�n, pro-embryogen�c masses are generated and these are also observed �n o�l palm.

It �s clear that for the trans�t�on of somat�c cells to cells that are embryogen�cally competent (de Jong et al., 1993), proper aux�n treatment �s needed, both �n terms of concentrat�on and durat�on of exposure (Dud�ts et al., 1991; Amm�rato, 1983). Now, the next quest�on �s why are only some cells capable of th�s trans�t�on? To add to th�s, �n the case of the o�l palm, both EC and NEC cultures can be der�ved and ma�nta�ned on an �dent�cal med�um w�th �dent�cal aux�n concentrat�on but yet take developmental d�fferent routes. The embryogen�c response �nduced by hormones �s h�ghly spec�es and genotype-spesific. A range in responses in tissue culture has been observed between genotypes, d�fferent reg�ons along the leaves and the phys�olog�cal state of the plant dur�ng sampl�ng (Borge et al., 1990; G Wong, pers. comm.).

The key �nducers of embryogen�c cell types �n the major�ty of t�ssue culture systems are aux�n treatment, typ�cally 2,4-D, and wound�ng. Therefore, aux�n- and wound-respons�ve genes are pr�mary cand�dates for the detect�on of changes �n the level of gene express�on. Borge et al. (1990) successfully used aux�n-respons�ve genes as molecular markers for detect�on d�fferences between embryogen�c and non-embryogen�c cultures of Medicago sativa cv. Rambler. The effects of aux�n (at the gene express�on level) can be detected as early as 2 to 3 m�n after treatment (McClure and Gu�lfoyle, 1987; Theolog�s et al., 1985). Abel and Theolog�s (1996) demonstrated that the aux�n �nduct�on of these early genes �s �ndependent of de novo prote�n synthes�s, �mply�ng that the �nd�cators necessary for the response are already present even before the add�t�on of aux�n. Hence, th�s response �s termed the pr�mary aux�n response by S�tbon and Perrot-Rechenmann (1997) who tabulated a l�st of aux�n up-regulated plant genes �n the same paper. Amongst the genes ment�oned are those �nvolved �n encod�ng for DNA-b�nd�ng prote�ns, an example be�ng the IAA (AUX) fam�ly wh�ch encode for short-l�fe nuclear prote�ns that conta�n a putat�ve


43

baa DNA-b�nd�ng mot�f (Abel et al., 1994). Bes�des th�s, genes encod�ng for other putat�ve DNA-b�nd�ng prote�ns have also been demonstrated to be up-regulated dur�ng aux�n treatment (Ba�ma et al., 1995; Hong et al., 1995a), re�nforc�ng the poss�b�l�ty of auxin influencing cellular activities through DNA-b�nd�ng prote�ns.

In add�t�on, genes encod�ng calc�um-modulated prote�ns that play an �mportant role �n ma�ntenance of cellular funct�ons �n plants (Irv�ng et al., 1992; Bush, 1993; Okamoto et al. 1995; Antos�ew�sc et al., 1995; Botella et al., 1996) as well as those �nvolved �n the regulat�on of cell d�v�s�on, e.g. cell cycle-assoc�ated genes, are also up-regulated by aux�n. It has been suggested that some components of the cell cycle are under hormonal control, and that the mRNA level of cdc2, �nvolved �n the G1-S and G2-M trans�t�ons of the cell cycle �n the form of prote�n k�nase p34cdc2, has been shown to be aux�n �nduc�ble (Mart�nez et al., 1992; Hemerly et al., 1993, John et al., 1993).

The role of aux�n has also been l�nked to the regulat�on of cell wall metabol�sm. Three classes of structural cell wall proteins identified as inducible by wound�ng and pathogen �nfect�on were also found to be up-regulated by aux�n �nduct�on (Ebener et al., 1993; Suzuk� et al., 1993; Vera et al., 1994). They are the hydroxyprol�ne-r�ch glycoprote�ns (HRGPs), glyc�ne-r�ch prote�ns and prol�ne-r�ch prote�ns (PRPs).

Can the changes �n these cell wall prote�ns be the cause of the �solat�on of cells dur�ng the format�on of pro-embryos or callus fr�ab�l�ty? Pennell (1998) stated that a cell identified by its wall can control the fate of another cell, and walls were found to be �nvolved �n cell-cell �nteract�ons suggest�ng that s�gnal molecules released from the walls are �nvolved �n these developmental processes �n the plant. Cells at d�fferent stages of the cell cycle were found to have character�st�c cell walls. Dolan et al. (1997) reported higher levels of unesterified pectins local�zed to non-d�v�d�ng cells than to d�v�d�ng cells �n the Arabidopsis root t�p. Moreover, HRGPs, extens�ns and arab�nogalactan-prote�ns (AGPs) were demonstrated to be dependent on the stage of the cell cycle and cell prol�ferat�on �n Catharanthus and carrot (Ho et al., 1998; Langhan and Nothnagel, 1997).

In embryogen�c suspens�on cultures of p�ne,there seems a h�gher accumulat�on of polysacchar�des, e.g. xylans, fucoxylans and arab�nogalactans, compared to �n non-embryogen�c l�nes (Mollard et al., 1997). Therefore, �t �s poss�ble that soluble AGPs can act as a s�gnal controll�ng somat�c embryogenes�s. Stud�es w�th monoclonal ant�body

JIM8 that recogn�zes a rhamnose-conta�n�ng ep�tope �n certa�n AGPs have demonstrated that cells identifiable by their wall composition release d�ffus�ble s�gnals, probably from the�r walls, to control developmental processes (McCabe et al., 1997). Bes�des AGPs, extens�n (a fam�ly of HRGPs) ep�topes were also assoc�ated w�th cell anatomy (Casero et al., 1998).

The aux�n-�nduced response of cell wall metabol�sm seems to m�m�c the responses by wound�ng and pathogen �nfect�on. Therefore, �t would be log�cal to assume that the funct�ons of these cell wall prote�ns dur�ng cell �solat�on �n embryogenes�s are �n wound heal�ng and the plant defense mechan�sm. In the case of pro-embryo format�on, �t �s poss�ble that when a st�mulus �s detected by the cell, there �s an �ncrease �n the secret�on of cell wall prote�ns �nto the wall, followed by rap�d �nsolub�l�zat�on �nduced. The convers�on of soluble prote�ns �nto the�r �nsoluble forms �s st�ll phenomenal. Epste�n and Lamport (1984) suggested that extens�ns may be cross-l�nked by �ntermolecular d�phenylether l�nkages or that extens�n monomers form a type of extens�n ol�gomer �n the presence of crude wall enzymes (Everdeen et al., 1988) although more recently, Bradley et al. (1992) has shown that extens�n �nsolub�l�zat�on �s enhanced w�th�n m�nutes of wound�ng, and el�c�tor or glutath�one treatment. It has also been hypothes�zed that th�s �nsolub�l�zat�on process �s med�ated by the release of hydrogen perox�de and �s catalyzed by a wall perox�dase. Interest�ngly, th�s seems to agree well with the physiological and molecular findings of th�s study. Once �nsolub�l�zat�on of the cell wall prote�n depos�t�on occurs, the cell wall �s rendered �mpenetrable (Showalter, 1993), �mped�ng, �n th�s case, cellular commun�cat�on between the pro-embryos and �ts ne�ghbour�ng cells. S�m�lar observat�ons have been made concern�ng callose depos�t�on. There �s st�ll a lack of �nformat�on regard�ng the funct�ons of AGPs, although �t �s bel�eved that they st�mulate non-embryogen�c cells to undergo embryogenes�s when secreted from the surface of embryogen�c cells (McCabe et al., 1997).

One of the earl�est aux�n-�nduced genes attr�buted w�th an enzymat�c funct�on �s the glutath�one-S-transferase (GST) fam�ly �n plants (Takahash� and Nagata, 1992). GSTs catalyse the transfer of reduced glutath�one to electroph�l�c substances (Hayes and Pulford, 1995; Marrs, 1996) thought to be involved in herbicide detoxification (T�mmerman, 1989). Moreover, GSTs are also �nvolved �n protect�on aga�nst ox�dat�ve damage. Th�s �nd�cates a connect�on w�th the new class of perox�dases, EgPER1 (Ong, 2001), �solated �n th�s study. It �s postulated that EgPER1 results from �nduct�on by aux�n prov�ded that the other necessary cond�t�ons are �n place.


44

It has been well demonstrated that aux�n �s respons�ble for regulat�on of a d�verse set of plant genes. And the rather large numbers of aux�n responsive genes reflect the variety of plant growth and developmental processes that �nvolve aux�ns. In somat�c embryogenes�s, Guzzo et al. (1994) suggested the poss�b�l�ty of the ex�stence of d�fferent classes of aux�n receptors. Thus, �f cells have or can be �nduced to produce the proper receptors, complete embryogenes�s would occur but �f the receptors are not correct, organogenes�s would be the preferred means of prol�ferat�on.

CONCLUSION

It �s obv�ous that there are many pathways �n the complex b�olog�cal system of plant development that can be tr�ggered �n t�ssue culture. Th�s �n�t�al aspect of the work �nvolv�ng anatom�cal and ultrastructural stud�es as well as some phys�olog�cal and b�ochem�cal exper�ments not prev�ously reported, have la�d the groundwork for future development of b�omarkers for embryogenes�s through gene express�on stud�es.

ACKNOWLEDGEMENTS

The authors would l�ke to thank the D�rector-General of MPOB for h�s support of the work descr�bed. Our heartfelt thanks to Appl�ed Agr�cultural Resources Sdn Bhd for prov�d�ng samples, the staff of the T�ssue Culture Plant Development Laboratory and the graduate students for the�r contr�but�ons to the research.

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Oil Palm Bulletin 53

Oil Palm Bulletin 53 (November 2006) p. 36 - 48 Biomarkers...

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